The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
A. Obesity
Obesity, which is defined as increased mass of adipose tissue, confers a higher risk of cardiovascular and metabolic disorders such as Type 2 diabetes, hyperlipidemia, and coronary heart disease and an associated morbidity and mortality. Metabolic syndrome, a multiplex risk factor for cardiovascular disease, is defined on the basis of five criteria relating to obesity, hypertriglyceridemia, low HDL cholesterol, high blood pressure and high fasting glucose (Grundy et al, Circulation (2004) 109:433-438).
Obesity is now a major healthcare issue in the Western World and increasingly in some third world countries. The increase in numbers of obese people is due largely to the increasing preference for high fat content foods but also, and this can be a more important factor, the decrease in activity in most people's lives. In the last 10 years there has been a 30% increase in the incidence of obesity in the USA and that about 30% of the population of the USA is now considered obese.
Whether someone is classified as overweight or obese is generally determined on the basis of their body mass index (BMI) which is calculated by dividing body weight (kg) by height squared (m2). Thus, the units of BMI are kg/m2 and it is possible to calculate the BMI range associated with minimum mortality in each decade of life. Overweight is defined as a BMI in the range 25.0-29.9 kg/m2, and obesity as a BMI of 30 kg/m2 or greater (see Table A below).
TABLE ACLASSIFICATION OF WEIGHTBY BODY MASS INDEX (BMI)BMICLASSIFICATION<18.5Underweight18.5-24.9Normal25.0-29.9Overweight30.0-34.9Obesity (Class I)35.0-39.9Obesity (Class II)>40  Extreme Obesity (Class III)
As the BMI increases there is an increased risk of death from a variety of causes that is independent of other risk factors. The most common diseases with obesity are cardiovascular disease (particularly hypertension), diabetes (obesity aggravates the development of diabetes), gallbladder disease, cancer and diseases of reproduction. Research has shown that even a modest reduction in body weight can correspond to a significant reduction in the risk of developing coronary heart disease.
There are problems however with the BMI definition in that it does not take into account the proportion of body mass that is muscle in relation to fat (adipose tissue). To account for this, obesity can also be defined on the basis of body fat content: greater than 25% in males and greater than 30% in females.
Obesity considerably increases the risk of developing cardiovascular diseases as well. Coronary insufficiency, atheromatous disease, and cardiac insufficiency are at the forefront of the cardiovascular complication induced by obesity. It is estimated that if the entire population had an ideal weight, the risk of coronary insufficiency would decrease by 25% and the risk of cardiac insufficiency and of cerebral vascular accidents by 35%. The incidence of coronary diseases is doubled in subjects less than 50 years of age who are 30% overweight. The diabetes patient faces a 30% reduced lifespan. After age 45, people with diabetes are about three times more likely than people without diabetes to have significant heart disease and up to five times more likely to have a stroke. These findings emphasize the inter-relations between risks factors for Type 2 diabetes and coronary heart disease and the potential value of an integrated approach to the prevention of these conditions based on the prevention of obesity (Perry, et al, BMJ (1995) 310:560-564).
Diabetes has also been implicated in the development of kidney disease, eye diseases and nervous-system problems. Kidney disease, also called nephropathy, occurs when the kidney's “filter mechanism” is damaged and protein leaks into urine in excessive amounts and eventually the kidney fails. Diabetes is also a leading cause of damage to the retina at the back of the eye and increases risk of cataracts and glaucoma. Finally, diabetes is associated with nerve damage, especially in the legs and feet, which interferes with the ability to sense pain and contributes to serious infections. Taken together, diabetes complications are one of the nation's leading causes of death.
The first line of treatment is to offer diet and life style advice to patients such as reducing the fat content of their diet and increasing their physical activity. However many patients find this difficult and need additional help from drug therapy to maintain results from these efforts.
Most currently marketed products have been unsuccessful as treatments for obesity owing to a lack of efficacy or unacceptable side-effect profiles. The most successful drug so far was the indirectly acting 5-hydroxytryptamine (5-HT) agonist d-fenfluramine (Redux™) but reports of cardiac valve defects in up to one third of patients led to its withdrawal by the FDA in 1998.
In addition, two drugs have recently been launched in the USA and Europe: Orlistat (Xenical™), a drug that prevents absorption of fat by the inhibition of pancreatic lipase, and Sibutramine (Reductil™), a 5-HT/noradrenaline re-uptake inhibitor. However, side effects associated with these products may limit their long-term utility. Treatment with Xenical™ is reported to induce gastrointestinal distress in some patients, while Sibutramine has been associated with raised blood pressure in some patients.
There is an unmet medical need for agents that safely decrease body weight. The present invention is directed to this, as well as other, important end.
B. Proopiomelanocortin (POMC) in Hypothalamus
The hypothalamic arcuate nucleus contains two discrete neuronal subsets, one that powerfully increases (orexigenic), and one that reduces (anorexigenic), food intake. The subset of neurons that increases food intake coexpresses two orexigenic molecules, neuropeptide-Y (NPY) and agouti-related peptide (AgRP). The subset of neurons that decreases food intake expresses proopiomelanocortin (POMC).
POMC is a prohormone from which are proteolytically derived in the hypothalamus a number of biologically active peptides, including but not limited to adrenocorticotropic hormone (ACTH), β-endorphin, α-MSH, β-MSH, and γ-melanocyte stimulating hormone (γ-MSH). Rodents lack the N-terminal proteolytic cleavage site for β-MSH, unlike most species, and are therefore β-MSH deficient. α-MSH and β-MSH bind to (and are agonists at) the melanocortin-4 receptor (MC4R) with a comparably high affinity, and γ-MSH binds exclusively to (and is an agonist at) the melanocortin-3 receptor (MC3R). α-MSH and β-MSH have been shown to be anorexigenic. Elevation of intracellular cAMP stimulates POMC expression and secretion of POMC-derived biologically active peptide. (Exemplary POMC mRNA and amino acid sequence can be found at GenBank® Accession No. BC065832 for human, GenBank® Accession No. BC061215 for mouse and GenBank® Accession No. BC058443 for rat.) (Exemplary NPY mRNA and amino acid sequence can be found at GenBank® Accession No. NM—000905 for human, GenBank® Accession No. NM—023456 for mouse and GenBank® Accession No. NM—012614 for rat.)
Humans and mice genetically lacking all POMC-derived peptides are severely obese. Humans and mice lacking the MC4R are markedly obese and hyperphagic. Codominant mutations of MC4R accounts for up to 5% of early-onset, severe obesity in humans and, as such, are the most common monogenic cause of severe obesity in humans. Recently, a missense mutation of β-MSH in humans has been shown to lead to early-onset, severe obesity and hyperphagia, features of MC4R deficiency.
ACTH and α-MSH have been shown to be antipyretic (to reduce fever, or pyrexia).
α-MSH, which is expressed not only in neurons but also in pituitary cells, keratinocytes and macrophages, and γ-MSH and other agonists at MC1R and at MC3R have been shown to be anti-inflammatory.
See, e.g., Pritchard et al, J Endocrinol (2002) 172:411-421; Barsh et al, Nature Reviews (2002) 3:589-600; Cone, Nature Neuroscience (2005) 8:571-578; Schwartz, Obesity (2006) 14:1S-8S; Yang et al, Brain Research (1996) 706:243-248; Boutillier et al, Pituitary (1998) 1:3343; Lee et al, Cell Metabolism (2006) 3:135-140; Biebermann et al, Cell Metabolism (2006) 3:141-146; Leibel, Cell Metabolism (2006) 3:79-81; Heneka et al, Peptides (2006) 114:8-18; Yoon et al, J Biol Chem (2003) 278:32914-32920; Murphy et al, Science (1983) 221:192-193; and Usui et al, Mol Cell Endocrinol (1989) 62:141-146.
C. GPR101
GPR101 is an orphan G protein-coupled receptor (GPCR) reported to be expressed in hypothalamus (Lee et al, Gene (2001) 275:183-91) and reported to elevate intracellular cAMP consistent with being coupled to Gs (WO 01/36471 A2). Expression of GPR101 in brain has been detected predominantly in discrete nuclei, including hypothalamus (Bates et al, Brain Res (2006)). The coding region for GPR101 is contained within a single exon.
D. G Protein-Coupled Receptors
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by the G protein-coupled receptor (GPCR) class. It is estimated that there are some 30,000-40,000 genes within the human genome, and of these, approximately 2% are estimated to code for GPCRs.
GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription pharmaceuticals have been developed. For example, in 1999, of the top 100 brand name prescription drugs, the following drugs interact with GPCRs (the primary diseases and/or disorders treated related to the drug is indicated in parentheses):
Claritin ® (allergies)Paxil ® (depression)Cozaar ® (hypertension)Propulsid ® (reflux disease)Pepcid ® (reflux)Effexor ® (depression)Allegra ® (allergies)Diprivan ® (anesthesia)Hytrin ® (hypertension)Plavix ® (MI/stroke)Xalatan ® (glaucoma)Harnal ® (prostatic hyperplasia)Prozac ® (depression)Zoloft ® (depression)Imitrex ® (migraine)Risperdal ® (schizophrenia)Gaster ® (ulcers)Depakote ® (epilepsy)Lupron ® (prostate cancer)BuSpar ® (anxiety)Wellbutrin ® (depression)Toprol-XL ® (hypertension)Singulair ® (asthma)Vasotec ® (hypertension)Zyprexa ®(psychotic disorder)Zantac ® (reflux)Serevent ® (asthma)Atrovent ® (bronchospasm)Cardura ®(prostatic hypertrophy)Zoladex ® (prostate cancer)Ventolin ® (bronchospasm)Zyrtec ® (rhinitis)Tenormin ® (angina)Diovan ® (hypertension)(Med Ad News 1999 Data).
GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transmembrane-3, transmembrane-4 and transmembrane-5, and transmembrane-6 and transmembrane-7 on the exterior, or “extracellular” side, of the cell membrane (these are referred to as “extracellular” regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on the interior, or “intracellular” side, of the cell membrane (these are referred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3), respectively). The “carboxy” (“C”) terminus of the receptor lies in the intracellular space within the cell, and the “amino” (“N”) terminus of the receptor lies in the extracellular space outside of the cell.
Generally, when a ligand binds with the receptor (often referred to as “activation” of the receptor), there is a change in the conformation of the receptor that facilitates coupling between the intracellular region and an intracellular “G-protein.” It has been reported that GPCRs are “promiscuous” with respect to G proteins, i.e., that a GPCR can interact with more than one G protein. See, Kenakin, Life Sciences (1988) 43:1095-1101. Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have been identified. Ligand-activated GPCR coupling with the G-protein initiates a signaling cascade process (referred to as “signal transduction”). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition. Although not wishing to be bound to theory, it is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
Gs-coupled GPCRs elevate intracellular cAMP levels. GPCRs coupled to Gi, Go, or Gz lower intracellular cAMP levels. Gq-coupled GPCRs elevate intracellular IP3 and Ca2+ levels.
There are also promiscuous G proteins, which appear to couple several classes of GPCRs to the phospholipase C pathway, such as G15 or G16 [Offermanns & Simon, J Biol Chem (1995) 270:15175-80], or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e.g. phospholipase C [Milligan & Rees, Trends in Pharmaceutical Sciences (1999) 20:118-24]. A GPCR coupled to the phospholipase C pathway elevates intracellular IP3 and Ca2+ levels.
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations: an “inactive” state and an “active” state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response. Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized in an active state by a ligand or a compound such as a drug. Recent discoveries, including but not exclusively limited to modifications to the amino acid sequence of the receptor, provide means other than ligands or drugs to promote and stabilize the receptor in the active state conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a ligand binding to the receptor. Stabilization by such ligand-independent means is termed “constitutive receptor activation.”